This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Radiation damage to biological samples is currently one of the major limiting factors in macromolecular x-ray crystallography. One of the best known method to reduce radiation damage at room temperature is flash freezing crystals to cryogenic temperature, which often requires finding penetrating cryoprotectants. Recently a new crystal freezing method, high pressure cryocooling, was developed, where using of cryoprotectants was avoided by freezing crystals under high pressures (1000 ~ 2000 atm). The mechanism was suggested involving high density amorphous (HDA) ice which has density of 1.17 g/cm3 at ambient pressure. Since the secondary radiation damage highly depends on the diffusion of the free radicals produced by x-ray radiation, HDA ice might be effective to reduce radiation damage due to its high viscosity and density compared to low density amorphous ice that usually forms inside crystal at ambient pressure. We will test the effect of high pressure cryocooling on radiation damage. For the study, high quality protein crystals will be prepared at several different pressures(1 atm ~ 4000 atm) and different chemical conditions.